The role of DDX3 in regulating Snail.

1Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Abstract

DDX3, a DEAD box protein family member, appears to promote the progression of some cancers, which may partly result from its impedance of death receptor-mediated apoptosis. We found that another mechanism by which DDX3 may aid cancer progression is by promoting increased levels of the transcription factor Snail. Snail represses expression of cellular adhesion proteins, leading to increased cell migration and metastasis of many types of cancer. Knockdown of DDX3 levels by shRNA reduced basal levels of Snail in HeLa and MCF-7 cells, and this was associated with reduced cell proliferation and migration. Snail protein and mRNA levels were increased by treatment with the HDAC inhibitors sodium butyrate or trichostatin A, and these increases were attenuated in cells with DDX3 knocked down. Treatment of cells with camptothecin was discovered to increase Snail protein levels, and this increase was diminished in cells with DDX3 knocked down. Analysis of 31 patient glioblastoma multiforme (GBM) samples revealed a significant correlation between the levels of DDX3 and Snail. Thus, DDX3 is required for basal Snail expression and increases in Snail induced by HDAC inhibitors or camptothecin, indicating that this action of DDX3 may contribute to its promotion of the progression of some cancers.

(A) E-cadherin, DDX3, Snail, and β-actin levels were immunoblotted in HeLa and MCF-7 wild-type (WT), DDX3 knockdown (KD), or mock knockdown (using a control shRNA) cells. Dark exposures of films were used to allow detection of low levels of Snail in cells with DDX3 knocked down. (B) DDX3 was knocked down by two different sequences of shRNA in MCF-7 cells, and DDX3 and Snail levels were immunoblotted. (C) Morphology of mock-shRNA (Control) and DDX3 knockdown (using clone #4 described in Figure 2B) MCF-7 cells. (D) Proliferation was measured in wild-type and DDX3 knockdown (KD; using clone #4 described in Figure 2B) HeLa cells (3000 cells), MDA-MB-231 cells (1000 cells), and MCF-7 cells (3000 cells; using clone #4 described in Figure 2B), along with a mock-shRNA control. Means ± SEM, n=3, *p < 0.05 compared with wild-type cells. E. Proliferation was measured in MCF-7 cells with control mock shRNA or clones #1 or #4 DDX3 knockdown described in Figure 2B. Means ± SEM, n=3, *p < 0.05. F. The wound healing assay was performed in mock-shRNA (Control) and DDX3 knockdown (using clone #4 described in Figure 2B) MCF-7 cells. The scratch was implemented and the cells were imaged immediately and after incubation for 2 days.

(A) MCF-7 cells were treated with 100 μM etoposide for 5-24 hr, and the levels of Snail, p53, and β-actin were immunoblotted. (B) MCF-7, (C) SH-SY5Y, or (D) MDA-MB-231 cells, which express mutant p53, were treated with 10 μM camptothecin for 2, 3, or 4 hr following incubation in serum-free media for 2 hr, and then Snail, p53, and β-actin levels were immunoblotted. (E) Cytosolic and nuclear p53 and Snail levels were measured after MCF-7 cells were treated with 10 μM camptothecin for 2, 3, or 4 hr. CREB was immunoblotted as a nuclear marker and α-tubulin as a cytosolic marker.

(A) Snail and 18S mRNA levels were measured in MCF-7 and SH-SY5Y cells after treatment with 10 μM camptothecin for 2, 3, or 4 hr. (B) Real time PCR was used to measure Snail and 18S mRNA levels in MCF-7 cells after treatment with 10 μM camptothecin. Means ± SEM, n=3. (C) H1299 p53-null cells were used, with or without inducibly expressing p53 for 24 hr, followed by treatment with 10 μM camptothecin for 2 or 4 hr, and Snail, p53, and β-actin levels were measured.

DDX3 knockdown-induced inhibition of camptothecin-induced Snail is a post-translational effect

(A) Mock-shRNA (Ctl) and DDX3 knockdown (KD) MCF-7 cells were treated with 10 μM camptothecin for 2 or 3 hr, and Snail, DDX3, and GSK3β in cell lysates were immunoblotted. (B) Snail and 18S mRNA levels were measured in mock-shRNA or DDX3 knockdown MCF-7 cells after treatment with 10 μM camptothecin for 2 or 3 hr. (C) Cytosolic and nuclear fractions were prepared from wild-type (WT) or DDX3 knockdown MCF-7 cells after treatment with or without leptomycin B (20 ng/ml) for 5 hr, and the levels of Snail, and DDX3 in both fractions were measured. Fractions were also immunoblotted with the cytosolic marker tubulin and the nuclear marker CREB. (D) Nuclear fractions were prepared from wild-type and DDX3 knocked down MCF-7 cells, DDX3 was immunoprecipitated, and DDX3 and Snail were immunoblotted.

Correlation between DDX3 and Snail levels in human glioblastoma samples

Snail and DDX3 levels were measured in 32 glioblastoma samples. Sample 15 was excluded from the analysis because of the absence of the β-actin loading control. The relative relationships between the expression of DDX3 and Snail were calculated as a percent of total human glioblastoma samples analyzed. Evaluation by the Chi-Square test demonstrated a significant correlation (p=0.001) between Snail and DDX3 levels.